Bottom Line:
Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2.However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation.Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.

ABSTRACTCell survival depends on proper propagation of protective signals through intracellular signaling intermediates. We report here that calponin homology domain-containing integrin-linked kinase (ILK)-binding protein (CH-ILKBP), a widely expressed adaptor protein localized at plasma membrane-actin junctions, is essential for transmission of survival signals. Cells that are depleted of CH-ILKBP undergo extensive apoptosis despite the presence of cell-extracellular matrix contacts and soluble growth factors. The activating phosphorylation of protein kinase B (PKB/Akt), a key regulator of apoptosis, is impaired in the absence of CH-ILKBP. Importantly, loss of CH-ILKBP prevents the membrane translocation of PKB/Akt. Furthermore, forced membrane targeting of PKB/Akt bypasses the requirement of CH-ILKBP for the activating phosphorylation of PKB/Akt, suggesting that CH-ILKBP is required for the membrane translocation but not the subsequent phosphorylation of PKB/Akt. Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2. However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation. Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.

fig4: Loss of CH-ILKBP impairs the membrane translocation of PKB/Akt. (A–E) The CH-ILKBP siRNA-transfectants (lanes 1 and 2), the control transfectants (lanes 3 and 4), and the parental HeLa cells (lanes 5 and 6) were serum starved (lanes 1, 3, and 5) or stimulated with 2 ng/ml IGF-1 for 5 min (lanes 2, 4, and 6). Samples from the cytosol (10 μg proteins/lane) and the membrane (corresponding to 20 μg cytosol proteins/lane) fractions were analyzed by Western blotting with antibodies recognizing Akt (A), phospho-Akt(Ser473) (B), phospho-Akt(Thr308) (C), CH-ILKBP (D), and PDK1 (E), respectively. (F) HeLa cells were transfected with the pUSEamp(+)/myr-Akt1 vector encoding myristoylated PKB/Akt (lanes 1–4) or a pUSEamp(+) vector lacking Akt sequence as a control (lanes 5–8). The myristoylated PKB/Akt and the control vector transfectants were then transfected with the CH-ILKBP siRNA (lanes 1, 2, 5, and 6) or the control RNA (lanes 3, 4, 7, and 8). The cell lysates (10 μg/lane) were analyzed by Western blotting with antibodies recognizing Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), and CH-ILKBP, respectively.

Mentions:
We next investigated the mechanism by which CH-ILKBP functions in the activating phosphorylation of PKB/Akt. Because membrane translocation of PKB/Akt precedes and is essential for the activating phosphorylation of PKB/Akt (Andjelkovic et al., 1997; Scheid et al., 2002), we tested whether CH-ILKBP is required for the membrane translocation of PKB/Akt. Stimulation of HeLa cells or the control cells with IGF-1 induced translocation of PKB/Akt from cytosol to the membrane (Fig. 4 A, lanes 4 and 6), where it was phosphorylated at Ser473 (Fig. 4 B, lanes 4 and 6) and Thr308 (Fig. 4 C, lanes 4 and 6). By marked contrast, the PKB/Akt membrane translocation (Fig. 4 A, lane 2) and phosphorylation at Ser473 (Fig. 4 B, lane 2) and Thr308 (Fig. 4 C, lane 2) were impaired in the absence of CH-ILKBP. Probing the samples with an anti–CH-ILKBP antibody confirmed that CH-ILKBP was depleted in the membrane and the cytosol fractions of the CH-ILKBP siRNA transfectants (Fig. 4 D, lanes 1 and 2). Loss of CH-ILKBP did not significantly alter the amount of PDK1 associated with the membrane (Fig. 4 E, lanes 1 and 2). These results indicate that CH-ILKBP is required for the membrane translocation of PKB/Akt but not the association of PDK1 with the membrane.

fig4: Loss of CH-ILKBP impairs the membrane translocation of PKB/Akt. (A–E) The CH-ILKBP siRNA-transfectants (lanes 1 and 2), the control transfectants (lanes 3 and 4), and the parental HeLa cells (lanes 5 and 6) were serum starved (lanes 1, 3, and 5) or stimulated with 2 ng/ml IGF-1 for 5 min (lanes 2, 4, and 6). Samples from the cytosol (10 μg proteins/lane) and the membrane (corresponding to 20 μg cytosol proteins/lane) fractions were analyzed by Western blotting with antibodies recognizing Akt (A), phospho-Akt(Ser473) (B), phospho-Akt(Thr308) (C), CH-ILKBP (D), and PDK1 (E), respectively. (F) HeLa cells were transfected with the pUSEamp(+)/myr-Akt1 vector encoding myristoylated PKB/Akt (lanes 1–4) or a pUSEamp(+) vector lacking Akt sequence as a control (lanes 5–8). The myristoylated PKB/Akt and the control vector transfectants were then transfected with the CH-ILKBP siRNA (lanes 1, 2, 5, and 6) or the control RNA (lanes 3, 4, 7, and 8). The cell lysates (10 μg/lane) were analyzed by Western blotting with antibodies recognizing Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), and CH-ILKBP, respectively.

Mentions:
We next investigated the mechanism by which CH-ILKBP functions in the activating phosphorylation of PKB/Akt. Because membrane translocation of PKB/Akt precedes and is essential for the activating phosphorylation of PKB/Akt (Andjelkovic et al., 1997; Scheid et al., 2002), we tested whether CH-ILKBP is required for the membrane translocation of PKB/Akt. Stimulation of HeLa cells or the control cells with IGF-1 induced translocation of PKB/Akt from cytosol to the membrane (Fig. 4 A, lanes 4 and 6), where it was phosphorylated at Ser473 (Fig. 4 B, lanes 4 and 6) and Thr308 (Fig. 4 C, lanes 4 and 6). By marked contrast, the PKB/Akt membrane translocation (Fig. 4 A, lane 2) and phosphorylation at Ser473 (Fig. 4 B, lane 2) and Thr308 (Fig. 4 C, lane 2) were impaired in the absence of CH-ILKBP. Probing the samples with an anti–CH-ILKBP antibody confirmed that CH-ILKBP was depleted in the membrane and the cytosol fractions of the CH-ILKBP siRNA transfectants (Fig. 4 D, lanes 1 and 2). Loss of CH-ILKBP did not significantly alter the amount of PDK1 associated with the membrane (Fig. 4 E, lanes 1 and 2). These results indicate that CH-ILKBP is required for the membrane translocation of PKB/Akt but not the association of PDK1 with the membrane.

Bottom Line:
Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2.However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation.Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.

ABSTRACTCell survival depends on proper propagation of protective signals through intracellular signaling intermediates. We report here that calponin homology domain-containing integrin-linked kinase (ILK)-binding protein (CH-ILKBP), a widely expressed adaptor protein localized at plasma membrane-actin junctions, is essential for transmission of survival signals. Cells that are depleted of CH-ILKBP undergo extensive apoptosis despite the presence of cell-extracellular matrix contacts and soluble growth factors. The activating phosphorylation of protein kinase B (PKB/Akt), a key regulator of apoptosis, is impaired in the absence of CH-ILKBP. Importantly, loss of CH-ILKBP prevents the membrane translocation of PKB/Akt. Furthermore, forced membrane targeting of PKB/Akt bypasses the requirement of CH-ILKBP for the activating phosphorylation of PKB/Akt, suggesting that CH-ILKBP is required for the membrane translocation but not the subsequent phosphorylation of PKB/Akt. Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2. However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation. Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.